Novel cysteine derivative

ABSTRACT

S-B-(4-pyridylethyl)-L-cysteine, a novel amino acid produced by reacting L-cysteine and 4-vinylpyridine in the presence of triethylamine, is ninhydrin-positive and very highly resistant to acid hydrolysis. It is particularly useful as an internal standard for chromotographic analyses of basic amino acids.

United States Patent Mendel Friedman;

James F. Cavlns, both of Peoria, Ill. 8,1 l 2 Jan. 2 l, 1970 InventorsAppl. No. Filed Division of Ser. No. 758,151, Sept. 6, 1968:

Patent No. 3,607,072

Sept. 2 1 197 1 The United States of Amerlca as represented by theSecretary of Agriculture Patented Assignee NOVEL CYSTEINE DERIVATIVE l Clalm, No Drawings U.S. Cl 23/230 R, 260/294.8 6

Int. Cl C07d 3l/48,

[50] Field olSeareh ..23/230,232 C; 260094.86

References Cited OTHER REFERENCES F. Kanne, Chem. Abstr. 59, 9300b(1963) Primary Examiner-Morris 0. Walk Assistant ExaminerR. M. ReeseAttorneys-R. Hoffman and W. Bier acids.

BACKGROUND OF THE INVENTION This invention relates to the 4ofa novelcysteine derivative, namely S-B-(4-pyridylethyl)-L-cysteine, thatexhibits a much more prolonged resistance to destruction by hydrochloricacid under the conditions of protein hydrolysis than do most of thepresently known amino acids, and that in addition also preciselyfulfills the linear ninhydrin and other requirements including discreteelution for use in amino acid analyses. Thus, the surprising fact thatS-B-(4-pyridylethyl)-L-cysteine is not destroyed by even 120 hours ofreflux in boiling HCI unexpectedly broadens its utility from that ofrare possible external standard for protein analysis to that of auniquely reliable internal standard for the analysis especially of basicamino acids, and which thereby enables the development of computerizedamino acid analysis systems that cannot be even contemplated with thecumbersome use of external reference standards nor only by the use ofnorleucine, which is well known to be operative only on acidic andneutral columns, i.e., for acidic and neutral amino acids.

Despite the well-known simplification and shortening of procedures thatare required when internal rather than external standards are used forvery precisely determining the concentrations of amino acids separatedby ion-exchange chromatography of protein hydrolysates, there previouslyhas been no amino acid that could serve as an internal standard on basicor neutral columns for basic or neutral amino acids similar to the useof norleucine primarily on acidic columns for acidic amino acids. Ournovel cysteine derivative is unique in that it can be used on basiccolumns as an effective internal standard for basic amino acids becauseit fulfills the following rigid requirements: (a) it isninhydrin-positive, and its ninhydrin color yield obeys Beers law; (b)it is extremely stable to acid hydrolysis in distinction to the proteinbeing digested; (c) it elutes in a position which does not overlap otheramino acid peaks; (d) and advantageously it is not a naturally occurringamino acid.

The present invention is directed to a novel derivative of L- cysteinewhich unobviously fulfills the above requirements and is, thus, areliable internal standard for extremely accurate amino acid analyses ofbasic amino acids.

One object of our invention is the provision of a novel amino acid thatis uniquely stable to acid hydrolysis, is linear as to its ninhydrinintensity, and which elutes in a position which is discernibly distantfrom the peaks of other basic amino acids.

A more specific object is the provision of a novel amino acid whosetotality of explicit properties make the new compound particularlyadvantageous as an internal standard for the calibration and calculationof amino acid analysis data obtained from ion-exchange chromatography ofacid hydrolysates of proteins.

GENERAL STATEMENT OF THE INVENTION In accordance with the herein statedobjects of the invention we have now synthesized the novel amino acidS-B-(4- pyridylethyl)-L-cysteine having the structure and havediscovered that our hydrochloric acid resistent new amino acid isuniquely suited for use as an internal standard for amino acid analysesby chromatography on basic columns by virtue of its acid resistance (100percent recovery at 24 hours and 98.4 percent at 120 hours), itsninhydrin color yield of 1.02%.03, and because it elutes from acidcolumns as a well-resolved peak completely subsequent to the histidineand ammonia peaks and completely prior to that of arginine.

Preparation of S-B-(4-pyridylethyl )-L-cysteine To a solution of g.(0.0165 mole) of L-cysteine in 40 ml. of deionized, distilled water wasadded L ml. (0.0l65 mole) of 4vinylpyridine and 2.3 ml. (0.0165 mole) oftriethylamine, and the reaction mixture was magnetically stirred undernitrogen atmosphere for 24 hours followed by successive reductions ofvolume and crystallizations in a rotary evaporator at 40 C. The totalweight of three crops of crystals was 4.9 g. percent). The structure ofthe compound, recrystallized from aqueous enthanol as fluffy needles,was confirmed by infrared nuclear magnetic resonance, and massspectroscopic analyses. The elemental analysis was as follows:

Anal. Calcd. for C,,,H,,N,SO, M22630); 1 G. 53.08; H, 6.24; N, 12.38; S,14.l7v Found: C, 53.02; N, 6.21; N, I24; 5, 14.!2.

EXAMPLE 2 The practical employment of the above obtained cysteinederivative was exhibited in the following manner wherein respectivelhydrolysates of bovine serum albumin and of Ponca flour in 2 ml.constant boiling 6N I-lCl also containing I00 gammas each ofS-B-(4-pyridylethyl)-L-cysteine for a basic column and of norleucine foran acidic or neutral column, were evaporated to dryness under reducedpressure. the residue redissolved in water, and after severalrepetitions finally redissolved in pH 2.2 citrate buffer for analysis.

The 3-hour procedure of Benson et al., Anal. Chem. 37:1l08 (i965 wasemployed using a Beckman Model l20 amino acid analyzer. Electronicquantitation of peaks on the basic column was based on our novelcysteine derivative whereas those on the acidic or neutral column werebased on the norleucine peak. The value equivalents for the two internal standard were calculated by a programmed digital computer fromseveral standard analyses using a known equation, and then theso-obtained equivalents were used in another equation to calculate themicromoles of the various amino acids per gram of sample.

Table I sets forth the thusly obtained percent amino acid compositionsof bovine serum albumin and of Ponca flour, which values are consistentwith the laboriously obtained literature values.

We claim:

I. In a chromatographic process for determining and preciselyidentifying and quantitating the basic amino acids in a hydrochloricacid-hydrolyzed protein material, the improvement comprising adding tothe protein material, prior to the said acid hydrolysis thereof, aninternal standard reference quantity of S-B-( 4-pyridethyl)-L-cysteine.

